Androgen action appears to occur only after conversion of testosterone (T) to dihydrotestosterone (DHT) or 5 alpha-androstanediol (3 alpha A) in some androgen responsive tissues. We postulate that DHT, glucuronide (DHT-G) and then to 3 alpha A-17 glucuronide (3 alpha A-17G) before it leaves the cell. Thus, plasma or urinary levels of specific 3 alpha A-17 glucuronide is the best in vivo marker of androgen action. To provide an efficient measure of these compounds, we propose to synthesize the nonradioactive and radioactive forms of testosterone glucuronide, DHT-G, 3 alpha A-17G,3 alpha A-3G and androsterone glucuronide and conjugate them to bovine thyroglobulin. These steroid-protein conjugates will be used to raise polyclonal or monoclonal antibodies to develop specific radioimmunoassays for each of the androgen conjugates. The conjugates will be measured in the plasma and urine of various subjects and correlated with androgen action. Direct experiments to demonstrate that the pathway, T to DHT to DHTG to 3 alpha A-17G, occurs in reproductive tissues and skin before 3 alpha A-17G is released into the plasma will be carried out in vivo using a dog model. The characteristics of the enzymes involved in this pathway, particularly the glucuronyl transferases, will be determined using standard in vitro biochemical techniques using tissues from the dog and skin biopsies from the human. These studies will lead to a better understanding of the of local androgen biosynthesis and metabolism in androgen action at the tissue level.